Structural variation in d(CTCTAGAG). Implications for protein-DNA interactions

Single-crystal X-ray diffraction techniques have been used to characterize the structure of the self-complementary DNA oligomer d(CTCTAGAG). The structure was refined to an R factor of 14.7% using data to 2.15-A resolution. The tetragonal unit cell, space group P4(3)2(1)2, has dimensions a = 42.53 and c = 24.33 A. The asymmetric unit consists of a single strand or four base pairs. Two strands, related by a crystallographic dyad axis, coil about each other to form a right-handed duplex. This octamer duplex has a mean helix rotation of 32 degrees, 11.3 base pairs per turn, an average rise of 3.1 A, C3'-endo furanose conformations, a shallow minor groove, and a deep major groove. Such averaged parameters suggest classification of the octamer as a member of the A-DNA family. However, the global parameters tend to mask variations in conformational parameters observed at the level of the base pairs. In particular, the central TpA (= TpA) step displays extensive interstrand purine-purine overlap and an unusual sugar-phosphate backbone conformation. These structural features may be directly related to certain sequence-specific protein-DNA interactions involving nucleases and repressors.     

Energetics of Z-DNA formation in poly d(A-T), poly d(G-C), and poly d(A-C) poly d(G-T).

The conformational change for the alternating purine-pyrimidine polydeoxyribonucleotides i.e. poly d(A-T), poly d(G-C), and poly d(A-C) poly d(G-T) from a right-handed conformation at room temperature to the left-handed Z-DNA like double helix at elevated temperatures has been studied by UV spectroscopy, Raman spectroscopy, and by adiabatic differential scanning microcalorimetry (DSC) in the presence of Na+ and Mg2+ or Ni2+ respectively as counterions. The differential UV spectra reveal through a hyperchromic shift at around 280nm and a hypochromic shift at 260nm that a conformational change to the left-handed conformation occurs. The Raman spectra clearly show characteristic changes, a drastic decrease of the band at 680cm-1 and the appearance of a new band at 628cm-1, due to the change of the purine bases to the syn conformation upon inversion of the helix-handedness. The course of the transition as function of temperature can be followed quantitatively by plotting the change in the excess heat capacity vs. temperature. The transition enthalpy delta H for the B- to Z-DNA transition per mole base pairs (mbp) amounts to 2.0 +/- 0.2kcal for poly d(G-C), to 4.0 +/- 0.4kcal for poly d(A-T), and to 3.1 +/- 0.3kcal for poly d(A-C) poly d(G-T). The enthalpy change due to the Z-DNA to coil transitions (per mole base pairs) amounts to 11kcal for poly d(G-C), 10.5kcal for poly d(A-T) and 11.3kcal for poly d(A-C) poly d(G-T).     

The post-SCF quantum chemistry characteristics of inter- and intra-strand stacking interactions in d(CpG) and d(GpC) steps found in B-DNA, A-DNA and Z-DNA crystals

The energies of intra- and inter-strand stacking interactions in model d(GpC) and d(CpG) two-base-pair steps were estimated by MP2/aug-cc-pVDZ single point calculations corrected for basis superposition errors. The stacked two-nucleobase pairs were constructed using experimental values of base pair and base step parameters taken from Nucleic Acid Database (). Three distinct polymorphic forms were analysed, namely A-, B- and Z-DNA. The applied methodology enables statistical analysis of structural and energetic diversities. The structural relationships between polymorphic forms are quite complex and depend on the sequence of pairs. The variability of parameters such as shift and tilt is almost the same irrespective of the polymorphic form and sequence of steps analysed. In contrast, shift and twist distributions easily discriminate all three polymorphic forms of DNA. Interestingly, despite significant structural diversities, the energies of the most frequent energy ranges are comparable irrespective of the polymorphic form and base sequence. There was observed compensation of inter- and intra-strand interactions, especially for d(GpC) and d(CpG) steps found in A- and B-DNA. Thus, among many other roles, these pairs act as a kind of energetic buffer, balancing the double helix. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Different forms of the double helix in the pCpGpCpGpCpG crystals

Four different crystal forms of the self-complementary oligonucleotide pCpGpCpGpCpG have been obtained. The space groups and the unit cell parameters of these crystals, as well as the location of the base-stacking reflections, indicate that the oligonucleotide is able to assume different double helical conformations that are dependent on the crystallization conditions.     

Effects of 5-fluorouracil/guanine wobble base pairs in Z-DNA: molecular and crystal structure of d(CGCGFG).

The chemotherapeutic agent 5-fluorouracil is a DNA base analogue which is known to incorporate into DNA in vivo. We have solved the structure of the oligonucleotide d(CGCGFG), where F is 5-fluorouracil (5FU). The DNA hexamer crystallizes in the Z-DNA conformation at two pH values with the 5FU forming a wobble base pair with guanine in both crystal forms. No evidence of the enol or ionized form of 5FU is found under either condition. The crystals diffracted X-rays to a resolution of 1.5 A and their structures have been refined to R-factors of 20.0% and 17.2%, respectively, for the pH = 7.0 and pH = 9.0 forms. By comparing this structure to that of d(CGCGCG) and d(CGCGTG), we were able to demonstrate that the backbone conformation of d(CGCGFG) is similar to that of the archetypal Z-DNA. The two F-G wobble base pairs in the duplex are structurally similar to the T-G base pairs both with respect to the DNA helix itself and its interactions with solvent molecules. In both cases water molecules associated with the wobble base pairs bridge between the bases and stabilize the structure. The fluorine in the 5FU base is hydrophobic and is not hydrogen bonded to any solvent molecules.     

Polarized electronic spectra of Z-DNA single crystals

Polarized electronic absorption spectra of the (100) face of single crystals of the Z-form double helical duplex of d(m5CGUAm5CG) have been obtained from Kramers-Kronig analysis of reflection data. The c crystallographic axis is parallel to the helix axis and shows but weak absorption. The b axis is perpendicular to the helix axis and shows a structureless absorption band centered at 270 nm with an oscillator strength of 0.26. Calculations of the crystal spectra utilizing available transition moment data for the individual chromophores are carried through using the oriented gas model (no interbase interactions) and, again, employing all base-base interactions (point dipole) in the duplex. The calculated hypochromism of the 270 nm band is much less than the experimental value obtained from the crystal data. The crystal spectra appear to be representative of Z-form double helices of essentially infinite length and not of a collection of twelve base duplexes. No evidence for n pi* transitions polarized parallel to the helix axis is found.     

The Structural and Functional Properties of Z-DNA

Biophysics - This review covers the stages of studying left-helical Z-DNA form, from its discovery in 1979 until the present time. The repetitive nucleotide sequences that are capable of the...     

A systematic study of ligand intermolecular interactions in crystals of copper(II) complexes of bidentate guanidino derivatives

The synthesis and X-ray structures of copper(II) complexes of the bidentate ligands, N-(4-oxo-5,5-diphenyl-4,5-dihydro-1H-imidazol-2-yl)-N′-phenylguanidino, 2-uanidinobenzimidazolo and N-(4-oxo-3-phenyl-1,3-diazaspiro[4.4]non-1-en-2-yl)guanidino, are reported. These complexes, which possess potential doublet (DA) or triplet (DAD) hydrogen bonding motifs, can form supramolecular structures based on synthons involving hydrogen bonding or phenyl embraces. The changes in supramolecular structure resulting from small changes in ligand structure, as well as from the use of different solvents for their crystallisation, are examined. The structures adopted are compared with others reported previously for complexes of related ligands.     

The high salt form of poly(dG-dC).poly(dG-dC) is left-handed Z-DNA: Raman spectra of crystals and solutions.

The laser-Raman spectra of crystalline d(CpGpCpGpCpG) and of aqueous poly(dG-dC).poly(dG-dC) in high salt (4M NaCl) and low salt (0.1M NaCl) solutions have been measured and compared. The spectra of the crystal and the high-salt solution show a striking congruence, which indicates clearly that the high-salt form of the aqueous polymer has the left-handed Z-DNA structure of the crystalline oligomer. These two spectra differ substantially from that of the low-salt form of the polymer, which has been found previously to have spectral characteristics of the B-form of DNA. The high salt spectrum shows a unique line due to guanine residues at 625 cm-1 which should be useful for qualitative and possibly quantitative assessment of the amount of Z-structure present in a sample of DNA.     

Probing intermolecular protein-protein interactions in the calcium-sensing receptor homodimer using bioluminescence resonance energy transfer (BRET).

The calcium-sensing receptor (CaR) belongs to family C of the G-protein coupled receptor superfamily. The receptor is believed to exist as a homodimer due to covalent and non-covalent interactions between the two amino terminal domains (ATDs). It is well established that agonist binding to family C receptors takes place at the ATD and that this causes the ATD dimer to twist. However, very little is known about the translation of the ATD dimer twist into G-protein coupling to the 7 transmembrane moieties (7TMs) of these receptor dimers. In this study we have attempted to delineate the agonist-induced intermolecular movements in the CaR homodimer using the new bioluminescence resonance energy transfer technique, BRET2, which is based on the transference of energy from Renilla luciferase (Rluc) to the green fluorescent protein mutant GFP2. We tagged CaR with Rluc and GFP2 at different intracellular locations. Stable and highly receptor-specific BRET signals were obtained in tsA cells transfected with Rluc- and GFP2-tagged CaRs under basal conditions, indicating that CaR is constitutively dimerized. However, the signals were not enhanced by the presence of agonist. These results could indicate that at least parts of the two 7TMs of the CaR homodimer are in close proximity in the inactivated state of the receptor and do not move much relative to one another upon agonist activation. However, we cannot exclude the possibility that the BRET technology is unable to register putative conformational changes in the CaR homodimer induced by agonist binding because of the bulk sizes of the Rluc and GFP2 molecules.     

Structure of poly d(A).poly d(T).

On the basis of the x-ray data from polycrystalline and well oriented fibers of the sodium salt of poly d(A).poly d(T) (Arnott et al, Nucl. Acids Res. 11, 4141-4155 (1983), a revised B'-DNA model incorporating B-like adenine and thymine strands is shown to give a much better x-ray agreement (R = 0.25) than the previously assigned model consisting of mixed sugar conformations in the two strands. The narrowing of the minor and the widening of the major grooves are promiscuous features of B'-DNA, which are common to all poly d(purine).poly d(pyrimidine) duplexes with two hydrogen bonded base-pairs and are in marked contrast with classical B-DNA. Due to modest propeller (-15 degrees), the cross strand diagonal hydrogen bonds (0.37 nm) in this duplex are not as strong as those in A,T-rich oligonucleotide crystal structures.     

G.T wobble base-pairing in Z-DNA at 1.0 A atomic resolution: the crystal structure of d(CGCGTG).

The DNA oligomer d(CGCGTG) crystallizes as a Z-DNA double helix containing two guanine-thymine base pair mismatches of the wobble type. The crystal diffracts to 1 A resolution and the structure has been solved and refined. At this resolution, a large amount of information is revealed about the organization of the water molecules in the lattice generally and more specifically around the wobble base pairs. By comparing this structure with the analogous high resolution structure of d(CGCGCG) we can visualize the structural changes as well as the reorganization of the solvent molecules associated with wobble base pairing. There is only a small distortion of the Z-DNA backbone resulting from introduction of the GT mismatched base pairs. The water molecules cluster around the wobble base pair taking up all of the hydrogen bonding capabilities of the bases due to wobble pairing. These bridging water molecules serve to stabilize the base-base interaction and, thus, may be generally important for base mispairing either in DNA or in RNA molecules.     

The octamers d(CGCGCGCG) and d(CGCATGCG) both crystallize as Z-DNA in the same hexagonal lattice

Both (dC-dG)₄ and d(CGCATGCG) crystallize in hexagonal lattices and their three-dimensional structure has been solved by x-ray diffraction analysis. Both molecules are found to form Z-DNA, although the fine details of the structure cannot be visualized due to the statistical disordering of the molecules along the c-axis, which is brought about by the symmetry constraints of the space group. This represents the first time in which the unmodified dinucleotide sequences CpAp and TpGp have been found to form Z-DNA in a crystalline lattice.     

Structural basis for Z-DNA binding and stabilization by the zebrafish Z-DNA dependent protein kinase PKZ

The RNA-dependent protein kinase PKR plays a central role in the antiviral defense of vertebrates by shutting down protein translation upon detection of viral dsRNA in the cytoplasm. In some teleost fish, PKZ, a homolog of PKR, performs the same function, but surprisingly, instead of dsRNA binding domains, it harbors two Z-DNA/Z-RNA-binding domains belonging to the Zalpha domain family. Zalpha domains have also been found in other proteins, which have key roles in the regulation of interferon responses such as ADAR1 and DNA-dependent activator of IFN-regulatory factors (DAI) and in viral proteins involved in immune response evasion such as the poxviral E3L and the Cyprinid Herpesvirus 3 ORF112. The underlying mechanism of nucleic acids binding and stabilization by Zalpha domains is still unclear. Here, we present two crystal structures of the zebrafish PKZ Zalpha domain (DrZalpha^PKZ) in alternatively organized complexes with a (CG)₆ DNA oligonucleotide at 2 and 1.8 Å resolution. These structures reveal novel aspects of the Zalpha interaction with DNA, and they give insights on the arrangement of multiple Zalpha domains on DNA helices longer than the minimal binding site.     

Conformational and intermolecular interactions of proteins in amyloidosis

A conception on amyloidosis as a key factor of neuronal death in neurodegenerative diseases is stated. Experimental evidence is presented that amyloidosis is caused by alterations in the activity of a number of enzymes as well as conformational changes in pathogenic proteins. Arguments for amyloidosis as the universal biological mechanism of specific elimination of neurons showing changed metabolic and physiological status of cell differentiation are adduced. The final pattern of cell death seems to differ cardinally from that in both apoptosis and necrosis.     

Conformation and intermolecular interactions of carbohydrate chains

For consideration of their conformations and interactions, carbohydrate chains can conveniently be divided into 3 classes on the basis of their covalent structure; namely periodic (a), interrupted periodic (b), and aperiodic (c) types. In aqueous solution carbohydrate chains often exist as highly disordered random coils. Under appropriate conditions, however, polysaccharides of types (a) and (b) can adopt a variety of ordered conformations. Physical methods, and in particular optical rotation, circular dichroism, and nuclear magnetic resonance, provide sensitive probes for the study of the mechanism and specificity of these disorder-order transitions in aqueous solution. Intermolecular interactions between such polysaccharide chains arise from co-operative associations of long structurally regular regions which adopt the ordered conformations. For acidic polysaccharides these cooperative associations may involve alignment of extended ribbons with cations sandwhiched between them. In other systems the interactions involve double belices which may then aggregate further, and geometric “matching” of different polysaccharide chains can also occur. These ordered, associated regions are generally terminated by deviations from structural regularity or by “kinks” which prevent complete aggregation of the molecules. The complex carbohydrate chains which occur at the periphery of animal cells have very different, aperiodic structures and although their conformations are as yet poorly understood, preliminary indications are considered.     

Aspecific and specific intermolecular interactions in aqueous media

Aspecific as well as specific interactions involve the same noncovalent forces, consisting of Lifshitz-van der Waals, Lewis acid/base, electrostatic, and thermal or Brownian movement interactions. In vivo, aspecific interactions between, e.g., cells and/or biopolymers usually are repulsive, while specific interactions are always attractive. The differences between the two classes of interactions can be shown to lie in the fact that aspecifically interacting bodies are large, while specifically interacting sites are small, or have a small radius of curvature, and in the fact that aspecifically interacting surfaces are homogeneous, whereas specific sites have a heterogeneous composition.     

Cobalt hexammine induced tautomeric shift in Z-DNA: the structure of d(CGCGCA)*d(TGCGCG) in two crystal forms

We report here the crystal structure of the DNA hexamer duplex d(CGCGCA).d(TGCGCG) at 1.71 Å resolution. The crystals, in orthorhombic space group, were grown in the presence of cobalt hexammine, a known inducer of the left-handed Z form of DNA. The interaction of this ion with the DNA helix results in a change of the adenine base from the common amino tautomeric form to the imino tautomer. Consequently the A:T base pair is disrupted from the normal Watson–Crick base pairing to a ‘wobble’ like base pairing. This change is accommodated easily within the helix, and the helical parameters are those expected for Z-DNA. When the cobalt hexammine concentration is decreased slightly in the crystallization conditions, the duplex crystallizes in a different, hexagonal space group, with two hexamer duplexes in the asymmetric unit. One of these is situated on a crystallographic 6-fold screw axis, leading to disorder. The tautomeric shift is not observed in this space group. We show that the change in inter-helix interactions that lead to the two different space groups probably arise from the small decrease in ion concentration, and consequently disordered positions for the ion.     

Effects of poly[d(pGpT).d(pApC)] and poly[d(pCpG).d(pCpG)] repeats on homologous recombination in somatic cells.

Sequencing studies have shown that in somatic cells alternating runs of purines and pyrimidines are frequently associated with recombination crossover points. To test whether such sequences actually promote recombination, we have examined the effects of poly[d(pGpT).d(pApC)] and poly[d(pCpG).d(pCpG)] repeats on a homologous recombination event. The parental molecule used in this study, pSVLD, is capable of generating wild-type simian virus 40 DNA via recombination across two 751-base-pair regions of homology and has been described previously (Miller et al., Proc. Natl. Acad. Sci. USA 81:7534-7538, 1984). Single inserts of either a poly[d(pGpT).d(pApC)] repeat or a poly[d(pCpG).d(pCpG)] repeat were positioned adjacent to one region of homology in such a way that the recombination product, wild-type simian virus 40 DNA, could be formed only by recombination within the homologies and not by recombination across the alternating purine-pyrimidine repeats. We have found that upon transfection of test DNAs into simian cells, a poly[d(pCpG).d(pCpG)] repeat enhanced homologous recombination 10- to 15-fold, whereas a poly[d(pGpT).d(pApC)] repeat had less effect. These results are discussed in terms of the features of these repeats that might be responsible for promoting homologous recombination.